Human γD-crystallin complementary DNA (cDNA) was cloned into the pET-21a vector, and the Y134X mutant clone was generated by site-directed mutagenesis. Wild-type and mutant proteins were overexpressed in the BL21 DE3 pLysS cells of E. coli. Wild-type protein was purified from the soluble fraction using the ion exchange and gel filtration chromatography methods. Mutant protein was predominantly found in insoluble fraction and purified from inclusion bodies. The structure, stability, aggregational, and amyloid fibril formation properties of the mutant were compared to those of the wild type using the fluorescence and circular dichroism spectroscopy methods.
Loss of the fourth Greek key motif in human γD-crystallin affects the backbone conformation, alters the tryptophan micro-environment, and exposes a nonpolar hydrophobic core to the surface. Mutant is less stable and opens its Greek key motifs earlier with a concentration midpoint (CM) of unfolding curve of 1.5 M compared to the wild type human γD-crystallin (CM: 2.5 M). Mutant is capable of forming self-aggregates immediately in response to heating at 48.6 °C.
Loss of 39 amino acids in the fourth Greek key motif of human γD-crystallin affects the secondary and tertiary structures and exposes the hydrophobic residues to the solvent. These changes make the molecule less stable, resulting in the formation of light-scattering particles, which explains the importance of the fourth Greek key in the underlying mechanism of opacification and cataract.
■将人γD-晶状体蛋白互补DNA(cDNA)克隆到pET-21a载体中,和Y134X突变体克隆通过定点诱变产生。野生型和突变型蛋白在大肠杆菌的BL21DE3pLysS细胞中过表达。使用离子交换和凝胶过滤色谱法从可溶性级分纯化野生型蛋白。突变蛋白主要存在于不溶性部分中,并从包涵体中纯化。结构,稳定性,聚合,使用荧光和圆二色谱方法将突变体的淀粉样原纤维形成特性与野生型的特性进行了比较。
■人类γD-晶状体蛋白中第四个希腊关键基序的丢失会影响骨架构象,改变色氨酸微环境,并将非极性疏水核暴露于表面。与野生型人γD-晶状体蛋白(CM:2.5M)相比,突变体的稳定性较低,其希腊关键基序的打开浓度(CM)为1.5M。突变体能够响应于48.6°C的加热而立即形成自聚集体。
■人γD-晶状体蛋白的第四个希腊关键基序中39个氨基酸的损失影响二级和三级结构,并将疏水性残基暴露于溶剂中。这些变化使分子不稳定,导致光散射粒子的形成,这解释了第四把希腊钥匙在浑浊和白内障的潜在机制中的重要性。